1. Field of the Invention
This invention relates to a process for preparing glucose-1-phosphate using an immobilized enzyme, and, more particularly, to a process for preparing, by the use of an immobilized enzyme, glucose-1-phosphate, which is a compound produced at the initial stage of a series of glycolysis reactions and is useful as an antibacterial agent for pharmaceutical applications, an antitumor agent (a platinum complex), a heart disease curing agent (amine salt), and the like.
2. Description of the Background
Conventionally, various processes for preparing glucose-1-phosphate from an .alpha.-glucan (starch, glycogen, or the like) and orthophosphate using enzymatic catalytic action of a phosphorylase have been known. Examples are a method using glycogen as an .alpha.-glucan and rabbit muscle extract as an enzyme fluid [Cori et al; J. Biol. Chem., 121, 465 (1937)] and a method using potato juice as an enzyme fluid and starch as an .alpha.-glucan [C. S. Hanes; Proc. R. Soc., B129, 174 (1940)].
More specifically, glucose-1-phosphate is synthesized using a glucan and orthophosphate as substrates and acting a phosphorylase on the substrates. After the reaction, the enzyme is heat-treated to degenerate and solidify it, and then removed from the reaction system. Unreacted orthophosphate is converted into an insoluble salt such as Mg.sub.3 (PO.sub.4).sub.2, MgNH.sub.4 PO.sub.4, Ba.sub.3 (PO.sub.4).sub.2, or the like, and removed for disposal. The unreacted glucan is removed by the use of an ion-exchange resin or by re-precipitating from an alcohol.
In these methods, however, the reaction is carried out batch-wise, requiring removal of the enzyme each time the reaction completes in the batch process. This entails a significant cost disadvantage in the industrial production.
The use of an immobilized enzyme with a phosphorylase carried onto a carrier is one of the measures for resolving this problem.
One of the processes to synthesize glucose-1-phosphate using an immobilized phosphorylase was reported by S. D. Larroque et al [J. Appl. Biochem., 4, 133 (1982)]. The report proposes immobilization of phosphorylase by combining the phosphorylase through an ionic bond onto a weakly basic anion-exchange resin which is prepared by introducing a diethylaminoethyl group into cellulose, through a hydrophobic bond onto a resin prepared by introducing an octyl group into agarose, and through a covalent bond onto a agarose which is activated by CNBr. All of these methods for immobilizing the phosphorylase, however, provide only an insufficient absorption of phosphorylase so that the immobilized enzyme exhibits only poor capability to synthesize glucose-1-phosphate. In addition, the use of polysaccharide resins in these methods involves difficulties in handling the resins, for example, in regenerating the resins. The methods are also disadvantageous in view of the production cost.
The need has therefore existed for a process for preparing glucose-1-phosphate through a more efficient use of phosphorylase, eliminating the above-mentioned problems.
In view of this situation, the inventors has undertaken extensive studies on the reaction of .alpha.-glucan and orthophosphate in the presence of an immobilized phosphorylase, and on the immobilized phosphorylase to be employed, in particular, and found that a phosphorylase carried onto a specific type of a carrier are suitable for this reaction. This finding has led to the completion of this invention.